Abstract

Biochemical and physiological processes in plants are affected by gamma-irradiation, which causes significant changes in gene transcripts and expression. To identify the differentially expressed Arabidopsis genes in response to gamma-irradiation, we performed a microarray analysis with rosette leaves during the vegetative stage. Arabidopsis plants were exposed to a wide spectrum doses of gamma ray (100, 200, 300, 400, 800, 1200, 1600 or 2000 Gy) for 24 h. At the dose range from 100 to 400 Gy, irradiated plants were found to be shorter than controls after 8 days of irradiation, while doses over 800 Gy caused severe growth retardation. Therefore, 100 and 800 Gy were selected as adequate doses for microarray analysis to identify differentially expressed genes. Among the 20,993 genes used as microarray probes, a total number of 496 and 1,042 genes were up-regulated and down-regulated by gamma-irradiation, respectively (P < 0.05). We identified the characteristics of the genes that were up-and down-regulated fourfold higher genes by gamma irradiation according to The arabidopsis information resource gene ontology. To confirm the microarray results, we performed a northern blot and quantitative real-time PCR with several selected genes that had a large difference in expression after irradiation. In particular, genes associated with lipid transfer proteins, histones and transposons were down-regulated by 100 and/or 800 Gy of gamma irradiation. The expression patterns of selected genes were generally in agreement with the microarray results, although there were quantitative differences in the expression levels.

Electronic supplementary material

Introduction

Due to the limited supply of genetic resources, mutagenesis using chemical, physical and biological mutagens has been employed as a mean of crop improvement [1]. Physical mutagens including gamma-rays, ion beams, microbeams and cosmic rays have been widely used for this purpose because plants only need to be exposed to these mutagens for a short time, large populations can be irradiated simultaneously and a large number of mutation spectra can be used [2, 3, 4, 5]. For decades, many mutant varieties have been utilized worldwide [6]. The most frequently used mutagenesis method for the creation of novel mutants is irradiation. Recently, radiation-derived mutagenesis and genomic researches using model plants, such as Arabidopsis and rice, have been steadily increasing [7, 8, 9]. Gene profiling of gamma-irradiated Arabidopsis plants is currently being performed using microarray techniques. For example, it has been used to examine transcripts expression during acute-chronic irradiation, during reproductive stage and ATM-mediated responses [10, 11, 12, 13].

Ionizing radiation is completely non-specific in its molecular targets and the response to this type of radiation is random [14]. Ionizing radiation affects genome structure and differential gene expression direct by energy or indirect effects by radicals. For indirect effects, ionizing radiation results in the generation of free radicals and reactive oxygen species (ROS) by the hydrolysis of water molecules and causes oxidative stress in the plant cells. Free radicals can attack various plant cell components, resulting in the disorder of cellular mechanisms and genetic variation in the DNA or chromosome levels. Radiation can also cause massive chromosomal aberrations and DNA damages, including DNA single-strand breakages (SSBs), DNA double-strand breakages (DSBs) and apurinic/apyrimidinic site mutations in many living organisms [15, 16, 17]. Irradiation activates the transcription of genes that are responsible for DNA repair and induces DNA repair mechanisms, such as homologous recombination, non-homologous end joining, and single-strand annealing [18, 19, 20]. Mutated genes induced by ionizing radiation can cause deleterious effects for the plant survival and severe damage may lead to abnormal function and finally to the death of the plant. Though there are negative effects on the plant genome, the induced mutations have been used to identify the function of mutated genes or to improve plant characteristics.

Several studies have shown that gamma-irradiation induces physiological changes in plants, such as root hair elongation, accumulation of anthocyanin and sucrose, induction of trichome formation, enhancement of respiration rate, expansion of root cell layers and cell wall damage [21, 22, 23, 24]. Such plant responses after irradiation can provide the standard in dose rate for mutation research with plant.

Arabidopsis thaliana is used extensively as a model plant for gene expressional profiling and functional analysis because it is well annotated and fully sequenced [25, 26]. It has many other advantages, such as a small genome size, short life cycle, prolific seed production, and diverse transgenic mutants. Among the methods for gene expression analysis, the microarray technique has been widely used for characterizing overall gene expression on a genomic scale. Microarray techniques allow for the estimation of transcript abundance for many plant organisms [27, 28]. In addition to transcript analysis, microarrays can be used for promoter mapping, polymorphism mapping, reverse sequencing, comparative genomic hybridization and whole genome analysis [29, 30, 31, 32]. The microarray analysis, however, is not reliable because of the reproducibility and compatibility. Therefore, after pre-screening of the transcript profiling by microarray, other experimental technique need to be performed to validate the microarray results.

Understanding the transcript variation generated by gamma-irradiation is essential for the characterization of unknown gene functions and for research into the plant radiation response mechanisms. Although there has been some research regarding specific genes that are induced by irradiation, the details of gene expression and the signaling cascades involved in plant responses to ionizing radiation still need to be elucidated [33, 34, 35].

In this study, A.thaliana was exposed to wide spectrum doses of gamma-ray during the vegetative stage, and we profiled the transcripts of Arabidopsis for genes that were up- and down-regulated by irradiation doses of 100 and 800 Gy. It is important to analyze the function of the gamma-irradiation responsive genes, not only for a further understanding of the mutation mechanisms of the DNA modifications and chromosome mutations of higher plants but also for the understanding of the signal perception and following response in plant after irradiation. We identified specifically up- or down-regulated genes by gamma-irradiation and validated by northern blot analysis and quantitative real-time PCR (qRT-PCR) with selected genes. These results may contribute to an overall understanding of the Arabidopsis genes involved during the vegetative stage whose expression is induced by gamma-irradiation.

Materials and methods

Plant growth condition and gamma irradiation

Arabidopsis (A. thaliana) ecotype Landsberg erecta was grown in autoclaved soil (1:1:1, v/v/v, mixture of vermiculite, perlite and peat moss) at 22 °C with a daily cycle of 16 h light and 8 h darkness. To test the radio-sensitivity of Arabidopsis plants during the vegetative stage, plants that had fully expanded rosette leaves were independently irradiated 18 days after seeding (DAS). Plants were exposed to 100, 200, 300, 400, 800, 1200, 1600 or 2000 Gy gamma-rays for 24 h. The source of ionizing radiation was 60Co with a half-life of 5.26 years (150 TBq of capacity; Nordion, Canada). The irradiated rosette leaves were collected and immediately frozen in liquid nitrogen until the RNA extraction 2 days after irradiation (DAI). A phenotypic evaluation of the growth performance was conducted at 8 and 23 DAI.

Microarray analysis

We used the Arabidopsis 2 Oligo (60-mers) Microarray Kit (V2) (Agilent, CA, USA) representing 21,500 Arabidopsis transcripts. The microarray experiments were conducted using the manufacturer’s recommended protocol. In brief, 1 μg of total RNA was labeled using the Agilent Fluorescent Linear Amplification Kit (Agilent). The mRNA was reverse-transcribed to cRNA and labeled with fluorescent dye. The control samples were labeled with Cy3 (green color) and irradiated samples labeled with Cy5 (red color) simultaneously hybridized to an array containing spots of oligo-sequences. The labeled target RNA was purified using the Qiagen RNeasy Mini Kit (QIAGEN, CA, USA). The labeled cRNA was resuspended in 400 μl of hybridization buffer (Agilent) and hybridized at 65 °C for 17 h. The hybridized microarrays were washed using the in situ Hybridization Kit Plus (Agilent). We used the Axon 4000B Scanner (Axon Instruments, CA, USA) to visualize the microarray slides. The microarray analyses were performed in duplicate with cRNAs that were prepared independently for each treatment.

Data extraction and analysis

The hybridized images were analyzed using the GenePix 6.0 (Axon Instruments). LOWESS and GeneSpring 7.3 (Agilent) were used for the data normalization and analysis, respectively. Significant genes were defined as genes with twofold change or greater compared to the expression baseline. The abnormal spots were excluded from further analyses. The changes in expression were calculated by dividing the median of the normalized red channel intensity by the median of the normalized green channel intensity. After normalization, we selected a total of 20,993 genes as microarray probes.

RNA isolation and northern blot analysis

The total RNA was isolated by the previously described method [36]. To validate the microarray results, high differentially expressed genes after different doses of gamma-irradiation were selected and subjected to northern blot analysis. The total RNA (15 μg) isolated from each sample was loaded onto 1 % (w/v) agarose gels containing 15 % (v/v) formaldehyde and blotted onto a Hybond N nylon membrane (GE Healthcare, Buckinghamshire, UK). The probes for the blotted genes were obtained by PCR using cDNA that was prepared from gamma-irradiated Arabidopsis plants. The probes were labeled with [α32-P]-dCTP using a Rediprime II Random Prime Labeling System (GE Healthcare) according to the manufacturer’s instructions. The gene specific primers used in this study are listed in Online Resource 2. The blot was hybridized in a Rapid-Hybrid buffer (GE Healthcare, UK) overnight at 65 °C, and the hybridized membranes were washed with 2× SSC plus 0.1 % (v/v) SDS buffer at 65 °C followed by washing with 0.2× SSC plus 0.1 % (v/v) SDS buffer. The X-ray films were exposed at −80 °C and the X-ray development was performed manually.

Gene ontology

To identify the characteristics of the up- and down-regulated genes after gamma-irradiation, we used The Arabidopsis Information Resource (TAIR) Gene Ontology (GO) to obtain the annotations, molecular functions, cellular components and biological processes of the genes. The gene classification was based on searches performed using BioCarta (http://www.biocarta.com/), DAVID (http://david.abcc.ncifcrf.gov/), and Medline databases (http://www.ncbi.nlm.nih.gov/). We categorized the radiation-responsive genes into six groups: up-regulated by 100 Gy irradiation, up-regulated by both 100 Gy and 800 Gy irradiation, up-regulated by 800 Gy irradiation, down-regulated by 100 Gy irradiation, down-regulated by both 100 and 800 Gy irradiation, and down-regulated by 800 Gy irradiation.

TaqMan probe-based qRT-PCR

The qRT-PCR was performed on an Eco Real-Time PCR System (Illumina, CA, USA) using the QuantiMix Probe One-Step Kit (Philekorea Technology, Deajeon, Korea) according to the manufacturer’s instructions. Each PCR amplification (20 μl of final volume) contained 250 nM of forward and reverse transcript-specific primers, 2× QuantiMix Probe One-Step Mixture, 0.2 units of RNase inhibitor, and 300 ng of total RNA. The PCR was initiated with 1 cycle of 42 °C for 15 min and 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s and 60 °C for 45 s. For each gene, three biological replicates were used and normalized for the average expression of the Arabidopsis actin gene. The primers had a melting temperature of approximately 58–60 °C. The Tm of the probe was approximately 10 °C higher than that of the primers. The gene specific primers used in this study are listed in Online Resource 3.

Results and discussion

Plant growth after a wide spectrum of gamma-irradiation and the determination of irradiation dose for microarray analysis

To identify the radio-sensitivity of Arabidopsis plant, we irradiated the vegetative stage plant with a wide spectrum of gamma-irradiation (100, 200, 300, 400, 800, 1200, 1600 or 2000 Gy) for 24 h (Fig. 1a). Eight days after irradiation with 100 Gy, these plants showed mild growth retardation compared to the control plants. The growth of plants irradiated with 200 Gy was more retarded than plants irradiated with 100 Gy, and the 200 Gy-irradiated plants grew to a height that was 50 % that of the control plants. At 8 DAI with doses over 400 Gy, these plants showed little evidence of growth and the ends of their leaves were rolled. In general, the plant growth patterns after irradiation showed that the negative effects on plant growth increased as the irradiation dose increased (Fig. 1b).

a Arabidopsis plants irradiated with gamma-rays at 100, 200, 300, 400, 800, 1200, 1600 and 2000 Gy. Plants were grown for 18 days after seeding and were irradiated for 24 h with each dose. b Plants 8 days after irradiation at each dose. c Plants 23 days after irradiation at 100, 200 and 400 Gy

At 23 DAI, the plants exposed to 100 and 200 Gy irradiation had heights that were two-thirds that of the control plants. The silique formation of 200 Gy-irradiated plants was greatly delayed compared to that of the control and 100 Gy-irradiated plants. The plants exposed to 400 Gy irradiation had fewer branches and silique formations than the plants exposed to doses of 200 Gy or lower. High doses of ionizing radiation (≥800 Gy) induced severe damage that could not be repaired during growth and development. (Fig. 1c). The height of fully mature plants exposed to doses of irradiation over 400 Gy was significantly reduced compared to that of the control plants and plants exposed to lower doses of irradiation [37]. The gross morphological appearance of a plant may change after irradiation: mild effects include a slight decrease in plant height and seedling vigor, while severe effects include a dramatic deviation from the non-irradiated control plants. In our study, plants irradiated with 100 and 800 Gy exhibited mild and severe effects, respectively. Given the plant responses to a wide range of radiation doses, we determined that 100 and 800 Gy were adequate doses for the microarray analysis.

Irradiated plants at low doses over a long period of time are more likely to generate useful and viable mutants than would acute irradiation [38, 39]. Arabidopsis seedlings exposed to low-dose gamma-rays (1–2 Gy) exhibited slightly higher growth rates than the controls [40]. A low dose of gamma-irradiation can have bio-positive effects on plant growth, which can be explained by “Hormesis” [41]. Hormesis is dose-dependent phenomenon which is characterized as plant response after irradiation: high-dose inhibition, low-dose stimulation in plant growth and development. Plant exposure to chronic radiation produces different responses and influences on plant morphology compared to acute radiation. Therefore, gene expression analysis using different chronic radiation dose is very useful for understanding the plant mechanisms and the signal cascades activated by irradiation perception. Kovalchuk et al. [12] reported that plants exposed to acute radiation in similar way as it responds to other stresses such as Cd, Pd, bleomycin and UVC. In contrast, the plant exposure to chronic irradiation was rather unique and had no similarities to any other stress conditions. It means that there was a definitely different response mechanism for plant exposed to acute or chronic radiation.

In addition, mutants with specific phenotypes after gamma irradiation will be very useful in understanding the influence after irradiation. Increased or decreased sensitivity for gamma rays would be helpful in understanding how biological and physiological processes in plant were affected by radiation stress.

Up-regulated and down-regulated genes under different doses of gamma-irradiation

Using microarrays, we profiled the Arabidopsis transcripts that were up- and down-regulated by irradiation doses of 100 or 800 Gy.

We considered genes with significantly different expression after irradiation if their expression signals were twofold higher than in the control plants and if the P value associated with the difference was <0.05. The average fold change was calculated by dividing the microarray intensity of the irradiated samples with that of the control plants. Among the 20,993 microarray probes, a total of 496 genes were up-regulated and 1,042 genes were down-regulated in the gamma-irradiated samples relative to the non-irradiated controls. The complete details of the genes that significantly responded to 100 and 800 Gy irradiation are provided as supplemental data.

In Figs. 2a and 3a, the Venn diagrams indicate the total number of genes that were up- and down-regulated by the gamma-irradiation. A total of 241 genes were up-regulated when Arabidopsis was exposed to 100 Gy irradiation. The total number of genes down-regulated by 100 Gy irradiation (739) was much higher than that of the up-regulated genes. There were 158 genes that were commonly up-regulated by irradiation at both 100 and 800 Gy.

a The number of up-regulated genes with statistically significant twofold or greater changes after 100 or 800 Gy irradiation, as determined by a one-tailed t test (P < 0.05). Further information regarding these genes can be found in the supplemental Table 1. b RNA gel blot analyses for selected up-regulated genes. Each lane was loaded with 15 μg of total RNA from rosette leaves harvested 2 days after gamma irradiation. At the top of each blot is the average ratio of the duplicate microarrays. Region of a is the genes up-regulated only in 100 Gy, and b is those only in 800 Gy

a The number of down-regulated genes with statistically significant twofold or greater changes after 100 or 800 Gy irradiation, as determined by a one-tailed t test (P < 0.05). Further information regarding these genes can be found in the supplementary Table 1. b RNA gel blot analyses for selected down-regulated genes by gamma-irradiation. Each lane was loaded with 15 μg of total RNA from rosette leaves harvested 2 days after gamma irradiation. At the top of each blot is the average ratio of the duplicate microarrays. Region of c is the genes down-regulated only in 100 Gy, and d is those only in 800 Gy

A total number of 413 genes were up-regulated as a result of exposure to 800 Gy. A nearly equal number of genes (391) were found to be significantly down-regulated by the same dose exposure. A total of 88 genes were down-regulated by irradiation both at 100 and 800 Gy. It was found that the biological responses to 100 Gy irradiation are significantly different from those of 800 Gy irradiation.

As we previously mentioned, response to an ionizing radiation revealed characteristics of randomness and many questions remain unanswered. Little is known about the primary changes in a gene expression irradiated with different doses and other radiation sources. But, we assumed that there were signal perception and responsive mechanisms to an ionizing radiation in plants. To elucidate the mechanism of a plant gene action after irradiation, many related researches are needed in plant radio-biology.

An additional approach to identify the gamma irradiation signal in plants is examined through the relationship with biotic and abiotic stress signals. Plants required oxygen and water for the survival and production of energy. Most plant cells after irradiation produce a variety of ROS, which can attack nucleic acids or other cellular components in the cell. During photosynthesis, ROS such as superoxide radical and hydroxyl radical are generated in the plant cell. Oxidative damage by ROS is a general phenomenon when the plant is exposed to several stress conditions. Radiation damage to plant cells is also mediated through the interaction of free radical and ROS.

Gene annotation of radio-responsive genes

To identify the characteristics of the genes that were up- and down-regulated by gamma irradiation, we categorized them using TAIR GO into standard categories, such as molecular function, cellular component and biological process (Online Resource 1). For the gene annotation, we classified those genes with a fourfold or greater change in expression. Because the microarray expression data have drawbacks, such as low technical quality and reproducibility, the analysis of genes with a twofold and greater change may be misleading.

In the molecular function category, the majority of the genes involved in catalytic activity were up-(37 %) and down-regulated (28 %) (Fig. 4a, b). In biological systems, catalytic activities are performed by enzymes that are essential for normal life processes. The activity of these enzymes can be changed by altering the cellular environment in which the catalytic activity occurs. The structure of enzymes is affected by massive doses of irradiation, which can either induce or repress the original enzyme activity. The down-regulated genes can be grouped into other significant functional classes, including nucleotide binding, protein binding, and ion binding. Gene modification in response to irradiation may be attributable to malfunctions in the binding activity of macromolecules, such as proteins, nucleotides and/or ions. The modification of the binding activity may be affected by the down-regulation of the catalytic activity and the transcription regulator activity.

Genes with fourfold or greater changes after 100 or 800 Gy irradiation were classified the TAIR GO annotation (http://www.arabidopsis.org/tool/bulk/go/index.jsp). a The molecular functions of the up-regulated genes, b the molecular functions of the down-regulated genes, c the cellular components of the up-regulated genes, d the cellular components of the down-regulated genes, e the biological process of the up-regulated genes, f the biological process of the down-regulated genes

Figure 4c, d presents the venn diagram for genes in the cellular components category that exhibited a fourfold or greater change in expression. Membrane-related genes, including those of the endomembrane system, were significantly affected by gamma-irradiation. Radiation-induced membrane damage may significantly affect plant metabolism. Casarett [42] has reported that cellular macromolecular components, such as cell walls and membranes, are affected by ionizing radiation. Radiation causes DNA damages, including SSBs and DSBs in the DNA strands and radio-induced DNA breakage may generate membrane abnormalities [43]. The genes involved in the nucleus and nucleosomes were down-regulated. The nucleus contains most of the cell’s genetic materials, which are organized as DNA molecules in complex with a large variety of proteins, including histones, to form chromosomes.

In the biological process category, more than half of the up-regulated genes were associated with either metabolism (29 %) or response to stimuli (26 %) (Fig. 4e). A high proportion of the changes in the expression of genes associated with metabolism are caused by chemical changes in living organisms, including anabolism and catabolism. The metabolic processes include macromolecular processes, such as DNA damage, repair and replication, and protein synthesis and degradation. The overall cell growth and organ development are also included in this category. Of the up-regulated genes, 26 % were associated with the response to stimuli. Kim et al. [13] demonstrated that genes involved in the response to stress and stimuli were up-regulated in Arabidopsis plants during the reproductive stage. A large percentage of down-regulated genes are associated with metabolism (28 %) (Fig. 4f). The down-regulation of reproduction-related genes (4 %) was observed and may be due to plant growth halting during the vegetative stages. The more details of GO information are provided as supplemental data.

To confirm the differential expression of the genes identified using microarray analysis, we performed a northern blot analysis for five genes with a high fold change as indicated by the microarray analysis per each group a–d shown in Figs. 2 and 3. The RNA from the control and irradiated plants were hybridized with selected probes to confirm overall expression. Northern blot analysis provides both a confirmation of the microarray results and a characterization of the overall expression with wide ranges of irradiation doses. To characterize the putative genes that are likely to be involved in irradiation responses, several genes that were up-regulated and down-regulated in response to 100 or 800 Gy irradiation were selected for the northern blot analysis. The primer sequences are provided as supplemental data.

Figures 2b and 3b showed the representative results of the northern blot analyses of the high fold-change genes. A nodulin family protein (At2g16660) and protein kinase family protein (At5g24010) showed slightly different expression patterns in the northern blot data than those produced from the microarray data in the 100 Gy-irradiated sample. At doses over 400 Gy, the transcript expression levels were slightly induced compared to the controls and lower doses of irradiation. Irradiation doses between 100 and 800 Gy yielded increased transcript levels of a protochlorophyllide oxidoreductase B gene (At4g27440). The transcripts of a U-box domain-containing protein (At1g66160) and an alcohol dehydrogenase (At1g09500) were significantly up-regulated only by 800 Gy irradiation. A minor increase in the expression levels of these genes was also observed in the 100 Gy-irradiated plants. The expression pattern of the alcohol dehydrogenase (At1g09500) was markedly higher for the high-dose irradiated samples (>800 Gy) compared to the control and 100 Gy-irradiated plants. The transcript levels of these genes increased proportionally with the increases of the radiation doses, and the maximum expression level was observed in the 2,000 Gy-irradiated samples which was relatively the highest dose in this study.

The expression patterns were inversely proportional to the dose of radiation for the down-regulated genes. A C2 domain-containing protein (At3g61720) was among the genes that were down-regulated by only 100 Gy irradiation; however, its expression was up-regulated at 200 Gy irradiation and was down-regulated at 300 Gy irradiation. Genes that were only down-regulated by 800 Gy irradiation (At5g16250, At3g54560, At5g22430 and At2g25880) exhibited drastically lower expression levels in all irradiated conditions; their expression levels decreased proportionally with the increase of the radiation doses. Although the band intensities of the RNA gel blots were not identical to the fold change of the microarray analysis, they showed very similar expression patterns.

The reliability and accuracy of the microarrays are questionable due to the existence of different technologies and variable interpretation of the obtained data. There is a discrepancy in the interpretation of the relative expression derived from the microarray, including the normalization method, background fluorescence adjustment, and use of mismatch probes [44]. Recently, Kim et al. [45] reported that four genes (At2g30360, At4g19130, At4g22960 and At5g24280) showed the same expression pattern regardless of seedling and leaf after gamma-irradiation. In particular, At2g30360 showed the same up-regulated pattern in our experimental conditions (100 Gy: 2.04 and 800 Gy: 2.56) in this study. This gene was commonly up-regulated, although many factors were different from each experimental condition. Therefore, At2g30360 gene could be used as radio-inducible marker genes for the identifying whether a sample is irradiated or not. But, other three genes were observed in different expression patterns. It may be attributed to the difference between two experimental conditions such as used ecotype (Landsberg versus Columbia), irradiation dose (100 or 800 vs. 200 Gy), dose rate (4.1 or 33.3 vs. 50 Gy h−1), developmental stage (vegetative versus reproductive) and unpredictable stress conditions or environmental factors including plant growth conditions.

Validation of the down-regulated genes using qRT-PCR

Among the many quantification methods, qRT-PCR has been widely accepted as the standard for gene expression measurement [46]. In this study, we try to identify the general down-regulated expression pattern for the selected genes. Although there are minor differences in the relative expression, most of the expression patterns of the down-regulated genes were consistent with the fold change of the microarray and qRT-PCR relative expression values.

There are three remarkable features in the profiling of the down-regulated genes: the down-regulation of the lipid transfer proteins (LTP) gene family genes, core histone genes and transposons. TaqMan-probe based qRT-PCR experiments were performed to validate the microarray results. The qRT-PCR results confirmed that these genes were down-regulated at least a fourfold after 100 and 800 Gy gamma-irradiation.

The down-regulation in transcript abundance for nine LTP gene family members was observed in the microarray experiments after gamma-irradiation (Table 1). These results were mostly in agreement with an earlier study of the gene expression patterns at 2 or 24 h after 3 kGy gamma-irradiation of Arabidopsis. Nagata et al. [11] reported that gamma-irradiation up-regulated several signal transduction and metabolic genes. They also reported that the LTP gene family genes were highly repressed.

Table 1

Comparison of LTP family gene expression by microarray and qRT-PCR methods

a The fold change indicates the mean value for the duplicated microarray

b This value shows data for the relative expression compared to control levels for selected genes. Values are shown as the mean ± SEM

The LTPs have many putative functions such as cutin synthesis [47], the plant pathogen-defense mechanism [48], plant signaling [49] and somatic embryogenesis [50]. Plant LTPs are thought to participate in membrane biogenesis and are partially bound to membrane [51]. The LTPs are a ubiquitous protein family in higher plants whose definitive biological function has not been conclusively elucidated [52]. Another research field in which plant LTPs are involved is the study of the plant’s ability to modify its metabolism in response to abiotic stress. Some studies have shown that LTP family genes are up-regulated by environmental stresses, such as drought, cold and high-salt conditions [53, 54, 55]. Plants can perceive gamma-irradiation as a physical stress. These observations indicate that plants have different signal perception mechanisms for irradiation than for other environmental stresses. Although there were quantitative differences between the microarray and qRT-PCR results, the pattern of expression for the down-regulated genes was consistent between each method for all LTP family genes.

The histone-related genes were only down-regulated by 800 Gy irradiation (Table 2). Histones are the main protein component of chromatin, acting as spools around which DNA winds. The chromatin structure contributes to the regulation of DNA transcription and repair. Radiation affects the various states of transcriptional control and the ability of plant cells to turn genes on and off as needed. Among the effects of radiation, the core histone components such as H2A, H2B, H3 and H4 were down-regulated by 800 Gy irradiation.

Table 2

Comparison of histone gene expression by microarray and qRT-PCR methods

AGI No.

Description

Fold change for microarraya

Relative expression for qRT-PCRb

100 Gy

800 Gy

100 Gy

800 Gy

800 Gy only down-regulated genes

At3g54560

HTA11, a histone H2A protein.

–

0.13

–

0.33 ± 0

At5g59870

HTA6, a histone H2A protein

–

0.16

–

0.43 ± 0.07

At3g20670

HTA13, a histone H2A protein

–

0.16

–

0.67 ± 0.15

At1g09200

histone H3

–

0.19

–

0.36 ± 0.05

At5g65360

histone H3

–

0.21

–

0.79 ± 0

At5g59690

histone H4

–

0.23

–

0.08 ± 0.12

At5g22880

histone H2B, putative

–

0.24

–

0.58 ± 0.06

a The fold change indicates the mean value for the duplicated microarray

b This value shows data for the relative expression compared to control levels for selected genes. Values are shown as the mean ± SEM

The expressions of a few genes encoding transposons were down-regulated by 100 Gy irradiation (Table 3). Transposons, including retrotransposons, are ubiquitous components of many plant organisms, where they are often a major component of nuclear DNA. They can induce mutations by inserting themselves near or within genes [56, 57]. Some studies have suggested that transposable elements are activated in response to stress, including biotic stress, cell culture and wounding [58, 59]. McClintock [60] proposed that transposable elements are silenced during normal growth conditions and activated by stress, corresponding to the genome restructuring or evolutionary roles of transposable elements. The abundant retrotransposons in plants are likely to be exposed to relatively weak radiation (100 Gy) and may lose their jumping function through mutations. It seems that plants have adaptive strategies to overcome the deleterious effects caused by radiation.

Table 3

Comparison of transposon expression by microarray and qRT-PCR methods

AGI No.

Description

Fold change for microarraya

Relative expression for qRT-PCRb

100 Gy

800 Gy

100 Gy

800 Gy

100 Gy only down-regulated genes

At3g30400

Gypsy-like retrotransposon family (Athila)

0.20

–

1.01 ± 0.10

–

At3g32240

CACTA-like transposase family (Ptta/En/Spm)

0.21

–

0.89 ± 0.06

–

At2g28980

Non-LTR retrotransposon family (LINE)

0.21

–

0.67 ± 0.05

–

At3g31390

Gypsy-like retrotransposon family (Athila)

0.23

–

0.85 ± 0.02

–

a The fold change indicates the mean value for the duplicated microarray

b This value shows data for the relative expression compared to control levels for selected genes. Values are shown as the mean ± SEM

Notes

Acknowledgments

This work was supported by grants from the Korea Science and Engineering foundation (KOSEF) in the Ministry of Science, ICT and Future Planning (MSIP) and the Korea Atomic Energy Research Institute (KAERI).